It is said that there have been viruses since so early stage of birth of life and have been evolving while contributing to evolution of organisms. There are many viruses having RNA as a gene (RNA viruses), and 90% of plant viruses are RNA viruses.
Recently, a method for introducing genetically-engineered genes into mammalian cells (a transfection method for animal cells) has been being studied intensively. As the method for introducing genes, a method utilizing a virus (such as a retrovirus) as a vector (a virus vector) (called a virus method) in order to transfect animal cells is widely used due to its relatively high transfection efficiency. In such a method utilizing a virus vector, a host cell is infected with the virus vector (a recombinant virus), thereby introducing a gene of interest into the host cell, wherein the virus vector is produced by partially recombining the gene of the virus with the gene of interest to be introduced or a promoter which can function in the host cell, or the like. Thus, the host cell uptakes and expresses an exogenous gene (i.e. a foreign gene).
In the virus method, it is necessary to use a virus vector having a high infection efficiency in order to obtain a sufficient transfection efficiency. The infection efficiency depends on many factors in a virus and/or a host cell such as: an invasion efficiency of the virus into the cell; a replication efficiency of the virus in the cell (some cells cannot be used for some viruses: this replication efficiency is referred to also as tropism); an expression efficiency of the viral gene in the host cell (an incorporation property into a genome, the number of viral gene copies, and the like); and the like. In order to improve the infection efficiency, various measures such as selection of cell types, improvement of a vector, addition of a secondary factor such as a T-antigen, and the like have been devised. However, the improvement of the infection efficiency has not yet been achieved, and is the largest factor which prevents application of the virus vector as a multipurpose vector. When a highly infectious virus vector is used, the gene recombinant is more likely to leak to the outside of an experimental laboratory so as to affect the environment.
Thus, there is great need for a technique for improving the transfection efficiency by treating the host cell as necessary without enhancing the infectious capacity of the virus vector itself. Each of cells of plants, insects, invertebrates, and vertebrates has an immune system for suppressing infection of an RNA virus as a host defense mechanism of organisms (a bioregulation mechanism). Thus, when it is possible to artificially depress the immune function, it may be possible to further improve the transfection efficiency in the virus method.
Recently, it is a problem to resolve the mechanism of the immune system how an innate immunity (basic immunity) system of plant, insects, invertebrates, and vertebrates detects and prevents virus invasion. A bioregulation mechanism (such as production of antibodies, and onset against virus-infected cells by lymph cells (called cytotoxic T lymph cells (CTLs)) has been developed by an acquired immune system appeared in the vertebrates. However, in order that the bioregulation mechanism functions sufficiently, it is necessary to help of the innate immunity such as an antigen-presenting cell. With completion of Genome Projects in various organisms, molecules involved in the innate immunity systems critical to the host defense (infection control) mechanism against bacteria and viruses are being identified. It has not been clarified for a long time which molecule regulates the host defense mechanism against the viral infection according to the innate immunity system and how to regulate the host defense mechanism by the molecule in human. However, only recently, it is gradually clarified to analyze the host defense mechanism at molecular level.
An initial immune response against a virus or a bacteria has been conventionally considered to be non-specific. However, a receptor group called microbial receptors was identified, so that it was clarified that: an immunocompetent cell of the innate immunity system such as macrophage and a dendritic cell detects foreign substances entered via a receptor, induces release of cytokine and activates lymph cells by expression of sub-stimulating molecules.
A Toll-like receptor which recognizes various microbial components and transmits a danger signal into a host is one of the foregoing microbial receptors, and such Toll-like receptors exist in plants, insects, mammals, and the like regardless of kinds. The Toll-like receptor is a homologue of a membrane protein (Drosophia Toll) involved in both development and immunity of Drosophila. Eleven members of the Toll-like receptors are found in human, and twelve members of the Toll-like receptors are found in mice. These Toll-like receptors constitute a group of a receptor family called a Toll-like receptor family. The Toll-like receptor has been noticed as a microbial receptor recently, and it has been clarified that the Toll-like receptor is involved in recognition of various microbial components.
Further, recently, it has been clarified that: a Toll-like receptor 3 (TLR3) which is one (kind) of the Toll-like receptors recognizes double-stranded RNA so as to activate a nucleic factor κB (hereinafter, referred to as “NF-κB”) (L. Alexopoulou, A. C. Holt, R. Medzhitov, R. A. Flavell, Nature 413 (2001) 732-738). That is, it was found that the Toll-like receptor 3 is a receptor involved in a double-stranded RNA-mediated signaling.
While, in the immune response of animal cells, it is known that type I interferon (interferon-α or interferon-β) which is one (kind) of cytokines plays an important role in defending against viral infection. Thus, it is considered that it is possible to drop an immune function against various kinds of viruses, when it is possible to prevent production of the type I interferon. Further, it is known that: when fibroblasts are stimulated with poly-(inosinic acid:cytidylic acid) (hereinafter, referred to as “poly(I):poly(C)” which is a synthesis analog of a viral double-stranded RNA (double-stranded RNA produced by a virus), transcription of the type I interferon is induced.
However, it has not been clarified how the animal cells recognize the viral double-stranded RNA and which signaling pathway produces the type I interferon. It was not known that signaling pathways involving in the production of the type I interferon exist in a downstream of the human Toll-like receptor 3.